Efficient prime editing in mouse brain, liver and heart with dual AAVs

Jessie R Davis; Samagya Banskota; Jonathan M Levy; Gregory A Newby; Xiao Wang; Andrew V Anzalone; Andrew T Nelson; Peter J Chen; Andrew D Hennes; Meirui An; Heejin Roh; Peyton B Randolph; Kiran Musunuru; David R Liu

doi:10.1038/s41587-023-01758-z

Efficient prime editing in mouse brain, liver and heart with dual AAVs

Nat Biotechnol. 2024 Feb;42(2):253-264. doi: 10.1038/s41587-023-01758-z. Epub 2023 May 4.

Authors

Jessie R Davis^#^{1

2

3}, Samagya Banskota^#^{1

2

3}, Jonathan M Levy^{1

2

3}, Gregory A Newby^{1

2

3}, Xiao Wang^{4

5

6}, Andrew V Anzalone^{1

2

3}, Andrew T Nelson^{1

2

3}, Peter J Chen^{1

2

3}, Andrew D Hennes^{1

2

3}, Meirui An^{1

2

3}, Heejin Roh^{1

2

3}, Peyton B Randolph^{1

2

3}, Kiran Musunuru^{4

5

6}, David R Liu^{7

8

9}

Affiliations

¹ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA.
² Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.
³ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA.
⁴ Cardiovascular Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁵ Division of Cardiovascular Medicine, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁶ Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA.
⁷ Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of MIT and Harvard, Cambridge, MA, USA. drliu@fas.harvard.edu.
⁸ Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.
⁹ Howard Hughes Medical Institute, Harvard University, Cambridge, MA, USA. drliu@fas.harvard.edu.

^# Contributed equally.

Abstract

Realizing the promise of prime editing for the study and treatment of genetic disorders requires efficient methods for delivering prime editors (PEs) in vivo. Here we describe the identification of bottlenecks limiting adeno-associated virus (AAV)-mediated prime editing in vivo and the development of AAV-PE vectors with increased PE expression, prime editing guide RNA stability and modulation of DNA repair. The resulting dual-AAV systems, v1em and v3em PE-AAV, enable therapeutically relevant prime editing in mouse brain (up to 42% efficiency in cortex), liver (up to 46%) and heart (up to 11%). We apply these systems to install putative protective mutations in vivo for Alzheimer's disease in astrocytes and for coronary artery disease in hepatocytes. In vivo prime editing with v3em PE-AAV caused no detectable off-target effects or significant changes in liver enzymes or histology. Optimized PE-AAV systems support the highest unenriched levels of in vivo prime editing reported to date, facilitating the study and potential treatment of diseases with a genetic component.

MeSH terms

Animals
Brain
CRISPR-Cas Systems
Gene Editing* / methods
Hepatocytes / metabolism
Liver / metabolism
Mice
RNA, Guide, CRISPR-Cas Systems*

Substances

RNA, Guide, CRISPR-Cas Systems

Abstract

MeSH terms

Substances

Grants and funding